Metabolic Stability – Expected Phase I and Phase II and Soft and Hard Reactive Metabolites

This assay is used to determine the metabolic stability of a test compound incubated with human, rat, dog, primate or mouse liver microsomes and to detect expected Phase I, Phase II, and trapped reactive metabolites.

 Required from Customer
  • Test article: 20 mg of powder
  • Molecular mass (exact mass) of test compound and its salt form
  • MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.


  • Stability:
    • Table of % remaining of test compound at each time point
    • Calculated t1/2 of test compound and control
    • Calculated in vitro intrinsic clearance of test compound
  • Detection of expected Phase I metabolites:
    • Retention time, peak area and number of isobars detected for up to three major expected biotransformations per species
  • Detection of expected Phase II metabolites (glucuronidation)
    • Results of positive control (Nefazodone)
    • Indication if glucuronide conjugates were detected, including retention time and signal intensity of the conjugates
  • Detection of reactive metabolites
    • Results of positive control (Nefazodone)
    • If conjugates of reactive metabolites of test article were detected following tabulated results will be provided:
      • Accurate mass measurements of molecular ions of detected conjugates of reactive metabolites, retention times and signal(s) intensity.
      • Extracted Ion Chromatograms and Survey High Resolution Accurate Mass Spectra (HRAMS) of the detected conjugates in representative samples.


  • Test compound at 1 µM dissolved in liver microsomal reaction mixture

Assay System

  • Human liver microsomes:  0.5 mg/mL (mixed gender, ≥ 10 donors)
  • Mouse, rat, dog, or monkey liver microsomes:  0.5 mg/mL (male, ≥ 3 donors)
  • Reaction mixture:
    • Potassium phosphate buffer, pH 7.4: 100 mM
    • Magnesium Chloride:  5 mM
    • Cofactors:
      • Phase I Metabolism: NADPH 1mM
      • Phase II Metabolism: UDPGA 1mM
      • Reactive Metabolites: GSH 5 mM containing stable labeled GSH (SIL-GSH)
      • Reactive Metabolites: KCN 1 mM containing stable labeled KCN (1:1 KCN:SIL-KCN)
    • Positive controls:
      • Phase I Metabolism: Testosterone
      • Phase II Metabolism: Nefazodone
      • Reactive Metabolites: Nefazodone

Assay Conditions

  • The assay is run with a single incubation (N=1)
  • Stability, Phase I/Phase II Metabolism, and Reactive Metabolites
    • Incubate test compound (1 µM) at 37°C in buffer containing 0.5 mg/mL microsomal protein
    • Incubate solvent control in parallel, sampling at 60 minutes
    • Initiate the reaction by adding cofactors (NADPH, UDPGA, GSH, and KCN), sampling at 0, 10, 20, 30, and 60 minutes
    • Incubate positive control (5 µM testosterone) in parallel, sampling at 0, 10, 20, 30, and 60 minutes
    • Incubate positive control (50 µM Nefazodone) in parallel, sampling at 60 minutes

Assay QC

  • The control compounds, testosterone and nefazodone, are run in parallel assays to verify the activity of the CYPs
  • After the final time point, fluorimetry is used to confirm the addition of cofactors to the reaction mixture
  • T1/2 of controls must meet internal acceptance criteria


  1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
  2. This assay does not provide stability information of test compound in liver microsomes without cofactors.
  3. Percent remaining at each time point is calculated from the peak area response ratio of test compound (or control) divided by the peak area response ratio of the 0-minute sample.
  4. In vitro intrinsic clearance (CLint in-vitro) = k/P (mL/min·mg protein), where k is the elimination rate constant (min-1) and P is the protein concentration (mg/mL).